Increasing evidence suggests that CD4+ helper T-cells (Th) are critical for the induction of an effective antitumor response. However, intracellular tumor antigens are usually difficult to be processed and presented to MHC class-II, limiting the activation of critical CD4+ Th cells. In our recent study, a model intracellular tumor antigen, MAGE-3, was genetically linked to a leader sequence and to a DC targeting domain for receptor- mediated internalization for DC activation and antigen presentation. Superior antigen-specific CTL responses and antitumor activity were induced by transduced DCs expressing the sMAGE3-Fc fusion protein (retrogen). In this proposal, we will extend this preliminary study with a long-term goal of developing more potent tumor vaccines by activating both antigen-specific Th and CTL responses. We hypothesize that activation of CD4+ Th by immunization with Th epitopes derived from the same antigen or retrogens will provide cognate help to CTLs and, thus, may be an effective means to enhance the potency of current tumor vaccines. Specifically, we will compare retrogen with other MHC class II targeting approaches and test if CD4+ Th plays a critical role in the enhancement of anti-tumor immune responses by retrogen in mice. We will identify novel class-II-restricted epitopes in MAGE-3 and related tumor antigens by utilizing the ability of retrogens to class II antigen presentation and the availability of HLA class-II transgenic mice. Furthermore, we will generate HLA-A2 and DR3/4 double transgenic mice to test if activation of antigen-specific Th cells can enhance the potency of tumor vaccines. We will also test if Th epitopes from the same antigen are more potent than nonspecific Th stimuli and if retrogen- transduced DC can induce more potent antitumor CTL and Th responses than DC pulsed with both Th and CTL epitopes in humanized mice. This proposed study will determine if activation of antigen-specific Th via immunization with either Th peptides or retrogen can enhance CTL and antitumor responses and if retrogen-transduced DCs are more potent than DCs pulsed with both Th and CTL epitopes or recombinant proteins to induce antitumor immune responses.